Endothelial dysfunction contributes to microvascular and macrovascular complications of diabetes. A major defense of endothelial cells against vascular injury is endothelial nitric oxide synthase (eNOS), which generates nitric oxide (NO) in the presence of optimal concentrations of the substrate L-arginine and the cofactor tetrahydrobiopterin (BH4). Diabetic nephropathy (DN) is associated with a state of progressive NO deficiency due to eNOS dysfunction, a phenomenon referred to as uncoupling. eNOS dysfunction and decreased bioavailability of NO in the kidney have recently been reported in an experimental model of type 1 diabetes and the protective role of NO generation by eNOS in the kidney has been conclusively established by recent studies showing that eNOS knockout mice made diabetic develop advanced lesions and progressive DN. While these studies established a protective role for eNOS, the mechanism(s) by which eNOS is inactivated in DN are not clearly identified. Understanding the mechanisms of uncoupling of eNOS in type 1 diabetes is essential if the above observations are to be translated to clinical therapeutic regimens aimed at recoupling of eNOS, restoring NO production and decreasing reactive oxygen species (ROS) generation during the course of DN. Our preliminary data show that in isolated glomeruli, where endothelium and mesangium are intact, eNOS generates ROS and lesser amount of NO upon stimulation with high glucose concentration (HG) or angiotensin II (Ang II). eNOS is expressed in glomerular endothelial as well as in mesangial cells and the enzyme generates ROS and less NO after exposure of the cells to HG or Ang II. HG and Ang II also cause a decrease in dihydrofolate reductase and an increase in arginase I protein levels, two enzymes that regulate availability of BH4 and L-arginine, respectively. Ang II and HG result in cell hypertrophy and fibronectin expression in cultured renal cells through generation of ROS via the NAD(P)H oxidase isoenzyme Nox4. Inhibition of Nox4 oxidase using an antisense oligonucleotide therapy reduces diabetes-induced Nox4 expression, renal ROS generation, hypertrophy and fibronectin expression in type 1 diabetic rats. Our central hypothesis is that the ROS generated by Nox oxidases and specifically Nox4 play a pivotal role in eNOS uncoupling in the glomerular mesangium and endothelium and in the kidney in diabetes, thereby resulting not only in the elimination of the protective effect of eNOS, but also converting the enzyme to a phlogistic mediator that further enhances ROS generation. We propose that ROS generated by Nox4 or other Nox oxidases result in oxidation of cysteine residues in the zincthiolate cluster essential for the activity of eNOS, and or decrease the levels of the substrate L-arginine and the cofactor BH4. The role of these pathways in hypertrophy, fibrogenic cytokine expression extracellular matrix accumulation and nuclear factor-kappaB activation will be explored in vitro and in vivo.